The main objective of the proposed research is to develop a cell culture system to study the process of neoplastic transformation in epithelial cells from rat esophagus after treatment with N-nitrosamine compounds and 12-0-tetradecanoylphorbol-13-acetate (TPA). Esophageal epithelial cells will be grown from explants in primary cultures and identified by morphological (light and electron microscopy), cytochemical and immunofluorescence methods. Conditions for serial propagation of the epithelial cells will be developed from quantitative studies of the effects of various media, sera, growth factors, hormones, substrates, etc., on growth in primary culture and in clonal culture. The epithelial cells will be treated with several concentrations of the N-nitrosamine compounds: N-nitrosomethylbenzylamine, N-nitrosodiethylamine and N-methyl-N-nitro-N-nitrosoguanidine alone, and in combination with TPA. Treated and control cultures will be monitored for transformation by examining the cells for alterations in morphology, growth rate and karotype; the synthesis of plasminogen activator; the ability to grow in soft agar; and the ability to produce tumors after transplantation into appropriate hosts. These investigations should result in the development of normal, preneoplastic and neoplastic esophageal epithelial cell lines that will be used for further studies of the molecular events leading to neoplastic transformation.